The term chromatography embraces a family of closely related separation methods. The feature distinguishing chromatography from most other physical and chemical methods of separation is that two mutually immiscible phases are brought into contact wherein one phase is stationary and the other mobile. The sample mixture, introduced into the mobile phase, undergoes a series of interactions (partitions) many times before the stationary and mobile phases as it is being carried through the system by the mobile phase. Interactions exploit differences in the physical or chemical properties of the components in the sample. These differences govern the rate of migration of the individual components under the influence of a mobile phase moving through a column containing the stationary phase. Separated components emerge in the order of increasing interaction with the stationary phase. The least retarded component elutes first, the most strongly retained material elutes last. Separation is obtained when one component is retarded sufficiently to prevent overlap with the zone of an adjacent solute as sample components elute from the column.
One kind of chromatography, which is widely used for biotechnological applications, is affinity chromatography. More specifically, affinity chromatography is a highly specific mode of chromatography wherein molecular recognition process takes place between a biospecific ligand and a target substance by a principle of lock-key recognition, which is similar to the enzyme binding to a receptor. For a general review of the principles of affinity chromatography, see e.g. Wilchek, M., and Chaiken, I. 2000. An overview of affinity chromatography. Methods Mol. Biol. 147: 1-6.
One advantageous class of affinity ligands comprises amine groups or acid groups. In the manufacture of such ligands, the amine groups or acid groups are often coupled to solid supports via the formation of an amide bond trough the activation of the carboxylic acid. In both cases, the formation of this type of chemical bond is not very efficient and is resulting in side products and media with poor homogeneity. For example, immobilisation of a NHS ester-activated ligand to an amine-derivatised support is never fully complete and some non-reacted amine groups will remain present. The same problem appears for the immobilisation of amine containing ligands to an ester activated derivatised support, in this case not reacted acid groups are remaining in the final media. Capping of these groups is as well possible, but the final product will still remain heterogeneous.
Accordingly, in this field, there is a need of alternative methods for generation of novel affinity ligands as well as for preparation of separation media thereof. There is also a need of optimising the binding properties of already known affinity ligands.
Finally, Feist and Danna (“Sulfhydryl cellulose: A New Medium for Chromatography of Mercurated Polynucleotides”. Patricia L. Feist and Kathleen J. Danna, Biochemistry, 20(15), p. 4243-4246) have disclosed a process of preparing sulfhydryl cellulose, which process includes to mix amino ethyl cellulose with an N-acetylhomocysteine thiolactone.